Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast ...cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using
and
breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of β-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.
Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing ...in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the ...implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.
In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term ...skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability – over 12 weeks – with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5×10
4
cells/cm
2 on epidermal tissue quality equalled that of the standard density (2×10
5
cells/cm
2). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte–fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.
Epidermal Homeostasis in Long-Term Scaffold-Enforced Skin Equivalents Stark, Hans-Jürgen; Boehnke, Karsten; Mirancea, Nicolae ...
The Journal of investigative dermatology symposium proceedings,
September 2006, 2006-Sep, 2006-09-00, 20060901, Letnik:
11, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and ...apoptosis. The lack of appropriate experimental models has largely prevented a better understanding of the regulatory mechanisms controlling epidermal tissue homeostasis in human skin. Keratinocyte culture studies had revealed a strict dependency of regular epidermal differentiation on dermal interactions only accomplishable in three-dimensional skin models. As major drawbacks, conventional models, employing collagen hydrogels as dermal equivalents (DEs) exhibit a rather poor stability and limited lifespan. Here, we present an improved stabilized in vitro-model for long-term growth and differentiation of keratinocytes providing the basis for tissue homeostasis. Keratinocytes were grown on DEs reinforced by modified hyaluronic acid fibers (Hyalograft-3D) and colonized with skin fibroblasts, producing genuine dermis-type matrix. These skin equivalents (SEs) develop superior epidermal architecture with regular differentiation and ultrastructure. Critical aspects of differentiation, still unbalanced in early stages, are renormalized, most strikingly the coexpression of keratins K1/K10, downregulation of regeneration-associated keratins (K16), and restriction of K15 to the basal layer. The strict localization of integrins to basal cells underlining restored tissue polarity, the drop of keratinocyte growth rates towards physiological levels and the rapid formation of a mature basement membrane with abundant anchoring fibrils are altogether features fulfilling the criteria of tissue homeostasis. Therefore, these scaffold-based SEs not only allow for studying homeostasis control but also for the first time provide proper experimental conditions for establishing a stem cell niche in vitro.
Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label‐retaining cells (LRCs). ...Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6–10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen‐ and scaffold‐based OTCs, only the scaffold‐OTCs supported long‐term survival and regeneration. LRCs' short survival in collagen‐OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)‐2 and MMP‐14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP‐dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long‐term tissue regeneration.
Disclosure of potential conflicts of interest is found at the end of this article.
Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising ...clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.
•A wide range of sensitivity to abemaciclib is observed among Rb+ tumor cells•CDKN2A mutant cancers show only intermediate sensitivity to CDK4/6 inhibition•D-cyclin activating features are associated with highly sensitive cells•About 5% of endometrial cancers bear a stabilizing mutation in the CCND1 3′UTR
Gong et al. identify a subset of cancers highly sensitive to CDK4/6 inhibition, which are characterized by various genomic aberrations known to elevate D-cyclin levels but not by CDKN2A mutations. They also identify a recurrent CCND1 3′UTR mutation associated with increased CCND1 expression in endometrial cancer.
Skin, as the largest organ, has long been subject of excellent and pioneering studies on stem cells and their role in tissue regulation and tumor formation. In particular, intensive research on mouse ...skin, and here especially the hair follicle, has largely extended our knowledge. Surprisingly, human skin, although the most easily accessible tissue in man, is far less conceived with regard to its stem cells and their specific environment (the niche). In consequence, these features are as yet only insufficiently defined and it still has to be elucidated how insights in cutaneous stem cell biology gained in mice can be extrapolated to humans. In the last few years, human model systems such as humanized mice or in vitro organotypic cultures that support maintenance or reconstruction of human skin and long-term epidermal regeneration have been developed. These models allow lineage tracing experiments and can be modified by adopting genetically manipulated cell types. Accordingly, they represent proper tools for human stem cell research and will clearly help to improve our still incomplete understanding. Like normal skin, the non-melanoma skin cancers and their respective tumors have gained considerable interest in basic as well as in clinical research. Being the most frequent human tumors globally, basal cell carcinomas and cutaneous squamous cell carcinomas (SCCs) continue to increase in incidence and specifically SCCs predominate in immunosuppressed transplant recipients. This review intends to compile the present knowledge on keratinocyte stem cells and their niches in normal skin and skin carcinomas with a special focus on the human situation. In particular, the role of the microenvironment, the niche, is emphasized, promoting our view of the decisive importance of the niche as a key regulatory element for controlling position, fate and regenerative potential of the stem cell population both in healthy skin and in carcinomas.
Abstract
Mutations in the transforming growth factor-β (TGF-β) pathway occur frequently in colorectal cancers (CRC). TGF-β is integral for key cellular processes, including proliferation, migration, ...differentiation and apoptosis. TGF-β/SMAD4 controls the activation of the pathway. A previously described R361H mutation in SMAD4 has been correlated with decreased overall survival and is suspected to modulate chemoresistance. The aim of the present study is to generate CRISPR-engineered SMAD4R361H CRC organoids and to utilize them as in vitro and in vivo models to investigate the effect of SMAD4R361H mutation on drug response. We have established independent organoid (PDO) and matching patient-derived xenograft (PDX) models from 5 different regions of a chemo-naïve CRC. By targeted amplicon sequencing using a cancer hot spot gene panel, we identified the SMAD4R361H mutation in two of the 5 subpopulations, whereas three of the PDOs were SMAD4wt. Semi-automated compound screening revealed a strong resistance phenotype to afatinib, sapitinib and gefitinib as compared to the wild-type models in vitro. Similar drug response was observed in in vivo testing. To investigate the mechanism of drug resistance, we have engineered isogenic organoids (SMAD4R361H/CRISPR) from SMAD4wt organoids using CRISPR-Cas9 genome-editing. In brief, SMAD4wt organoids were transfected with an all-in-one spCas9-sgRNA-vector and a ssODN as repair template bearing the R361H point mutation. To increase homologous-directed repair, we co-transfected the organoids with a plasmid vector containing i53-bpA, an inhibitor of 53BP1. Single clones were then isolated and genotyped by locus specific PCR to confirm the particular R361H point mutation. We were able to achieve up to 50 % knock-in efficiency in these organoids. Utilizing CRISPR-Cas9, engineered SMAD4R361H/CRISPR organoids were established. These PDOs were subjected with both, native SMAD4wt and SMAD4R361H PDOs, to an in vitro drug screening, allowing for a direct comparison of their drug sensitivity. Based on these results, confirmatory in vivo experiments were designed to investigate how SMAD4 may be involved in modulating resistance in CRC organoids. These models have a prominent role in discovering the connection between genotype and phenotype in complex PDO and PDX models. While this approach is not limited to studying the role of the R361H mutation, it has a broad range of applications in understanding cancer biology, intra-tumor heterogeneity, drug response and may eventually lead to new clinical insights, including design of new therapeutic strategies.
Citation Format: Ulrike Pfohl, Alessandra Silvestri, Dirk Schumacher, Karsten Boehnke, Johannes Haybaeck, Sanam Bashir, Ralf Kühn, Marlen Keil, Reinhold Schäfer, Wolfgang Walther, Christian Regenbrecht. Modulating chemoresistance: Uncovering the role of mutant SMAD4R361H in colorectal cancer using PDO and PDX models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4525.
Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to ...mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity
and
, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.
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